Francesco De Matteis

Decreased levels of cytochrome P-450 and catalase in hepatic porphyria caused by substituted acetamides and barbiturates: Importance of the allyl group in the molecule of the active drugs

Abstract The effect of four substituted acetamides and of two barbiturates on the levels of cytochrome P-450 and catalase has been studied and related to the induction of porphyria in the liver of rats. Drugs containing one or two allyl substituents were found to depress the activity of catalase and the levels of cytochrome P-450; they were also porphyrogenic. The significance of these findings is discussed in the light of recent evidence for destruction of liver haem in experimental porphyria.

Oxidative metabolism of carbon disulphide by the rat: Effect of treatments which modify the liver toxicity of carbon disulphide

Abstract The liver toxicity of a standard dose of CS2 was increased by phenobarbitone pre-treatment and by fasting and decreased by prior administration of a small dose of CS2; it was less marked in female than in male rats. Rats were given an intraperitoneal dose of 14CS2 to study its in vivo conversion to 14CO2 under conditions which modify the liver toxicity of CS2. The radioactive CO2 exhaled was found to be linearly related to the amount of cytochrome P-450 present in the liver at the time of administering 14CS2; it was also found to be related to the severity of the toxic changes caused in the liver, but here the relationship was not linear and was more complex. When liver microsomes were incubated with CS2, a marked loss of cytochrome P-450 was observed only when NADPH was present. In contrast with the loss of the cytochrome observed with NADPH alone, which is associated with the formation of malonaldehyde, that due to CS2 still occurred in the presence of EDTA, even though the formation of malonaldehyde was largely prevented. These results are compatible with the hypothesis that CS2 requires metabolism for its liver toxicity.

The mechanism of the suicidal, reductive inactivation of microsomal cytochrome P-450 by carbon tetrachloride

1. Stoichiometric losses of microsomal haem and cytochrome P-450 were observed when carbon tetrachloride (CCl4) was incubated anaerobically with rat liver microsomes using NADPH or sodium dithionite as a reducing agent. A rapid destruction of haem was also observed during the non-enzymatic reductive incubation of CCl4 with soluble haem preparations (methaemalbumin) in presence of sodium dithionite. The results indicate that haem is both the site and the target of the suicidal activation of CCl4 by cytochrome P-450. 2. When an additional, fluorimetric assay for haem determination was used, an equimolar loss of protoporphyrin IX fluorescence was also observed in both the enzymatic and non-enzymatic system, indicating that the haem moiety of cytochrome P-450 has undergone a structural change, involving either loss or labilization of the porphyrin tetrapyrrolic structure. In both systems the loss of porphyrin was prevented by carbon monoxide (CO). 3. A dichlorocarbene-cytochrome P-450 ligand complex is partially responsible for the difference spectrum obtained on addition of CCl4 to anaerobically reduced rat liver microsomes. A molar extinction coefficient for this complex has been calculated. The carbene trapping agent 2,3-dimethyl-2-butene (DMB) strongly inhibited (greater than 95%) the formation of this spectrum but did not modify the loss of haem in reduced CCl4-supplemented microsomal incubations. The results suggest that dichlorocarbene (:CCl2) is not significantly involved in CCl4-dependent haem destruction. 4. Pretreatment of rats with different microsomal enzyme inducers was responsible for similar but not identical patterns of :CCl2 and CO formation and haem loss during incubation of CCl4 with reduced microsomes. This indicates a critical role of CCl4 metabolism in the suicidal destruction of cytochrome P-450 haem and suggests that the apoprotein of cytochrome P-450 is capable of modulating not only the metabolism of CCl4 to :CCl2 but also the hydrolysis of :CCl2 to CO. 5. Inactivation of cytochrome P-450 by CCl4 with reduced microsomes from Aroclor-pretreated rats was saturable and followed pseudo first-order kinetics. This provides further evidence to conclude that CCl4 activation is a suicidal process where the reactive metabolite(s) formed bind to haem, we predict, in a one to one stoichiometry. 6. The partition ratio between loss of cytochrome P-450 haem and CCl4 metabolism by liver microsomes from Aroclor pretreated rats has been investigated using limiting concentrations of CCl4. It was calculated that approximately 26 molecules of CCl4 had to be metabolised to achieve the loss of one molecule of haem.

Destruction of endogenous and exogenous haem by 2-allyl-2-isopropylacetamide: Role of the liver cytochrome p-450 which is inducible by phenobarbitone

Abstract 1. 1. The study of the kinetics of cytochrome P-450 loss caused by 2-allyl-2-isopropylacetamide in vivo showed the existence of at least two components: pre-treatment with phenobarbitone markedly increased the amount of the fast decaying component without altering its half-life, but did not change significantly either amount or half-life of the slow decaying component. 2. 2. It is suggested (i) that the two components represent different cytochromes which are destroyed at different rates by 2-allyl-2-isopropylacetamide; and (ii) that phenobarbitone pre-treatment stimulates the destruction of cytochrome P-450 by increasing the concentration in the microsomal fraction of the type of cytochrome which is susceptible to rapid destruction. 3. 3. Exogenous haem was also destroyed when the liver microsomal fraction was incubated with the drug in vitro . This suggests that the action of the drug is not confined to the haem of the preexisting cytochrome P-450 but may extend to other pools of haem in the liver.

The glutathione-linked metabolism of 2-allyl-2-isopropy-lacetamide in rats. Further evidence for the formation of a reactive metabolite

2-Allyl-2-isopropylacetamide (AIA) causes a depletion of liver glutathione in rats only if the animals have been pretreated with phenobarbitone. Phenobarbitone stimulates the excretion in bile of a component derived from AIA and glutathione which is apparently not the same as the conjugate formed by reaction of the two components in simple solutions. The significance of these findings are considered in relation to the suggestion that AIA is metabolised to an epoxide by the microsomal enzyme system; in addition several differences between AIA and the non-porphyrogenic compound, acrylamide, are discussed.

Lobes of rat liver respond at different rates to challenge by dietary hexachlorobenzene

Abstract The lobes of livers from rats fed hexachlorobenzene (HCB) develop porphyria at different rates. The caudate lobe reacts significantly slower than the median, although eventually all lobes become equally porphyric. Uroporphyrinogen decarboxylase and 5-aminolaevulinate synthetase activities are less depressed and less elevated respectively in the caudate lobe than the remainder of the liver. The variations in porphyrin levels do not appear to be due to differences in clearances of the porphyrins from the tissues. HCB levels in the lobes, although similar at the later stages of porphyria, are significantly different in the initial course of treatment. The slower reacting caudate lobe contains the highest concentrations of HCB as shown by both tissue analysis and [U-14C]HCB experiments. The differences in rate of porphyria development between median and caudate lobes cannot therefore be accounted for by differences in uptake of HCB but must reflect variations in metabolism of HCB and/or in the porphyrogenic response. Some of the possibilities have been investigated.

Copper-induced dealkylation studies of biologically N-alkylated porphyrins by fast atom bombardment mass spectrometry

Abstract Copper has been reported to promote the demethylation of N -methylporphyrins. Copper-induced dealkylation is observed at room temperature in acetonitrile in the presence of a suitable nucleophilic acceptor molecule, or in chloroform alone. Electronic absorption spectra of N -methylprotoporphyrin IX (in the form of its dimethyl ester) in the presence of copper (with either chloroform alone or acetonitrile-dibutylamine as solvent) exhibited a transient spectrum corresponding to a copper chelate of the N -methylprotoporphyrin followed by ejection of the N -methyl group to afford the copper chelate of protoporphyrin. The use of fast atom bombardment mass spectrometry (FAB-MS) to identify the N -alkyl group of a series of N -substituted porphyrins was investigated. The alkyl group released on reaction with copper ions was trapped by an amine (e.g., dodecylamine). The resulting mixture was analysed by positive-ion FAB-MS using 3-nitrobenzyl alcohol as matrix. Molecular ions (MH + ) were detected at m/z 200, 214 and 228, corresponding to [CH 3 (CH 2 ) 11 NHCH 3 + H] + , [CH 3 (CH 2 ) 11 NHC 2 H 5 + H] + and [CH 3 (CH 2 ) 11 NHC 3 H 7 + H] + , respectively. The analysis was extended to N -alkylprotoporphyrins isolated from liver homogenates of mice treated with the porphyria-inducing drug griseofulvin. Using a combination of UV and copper-induced dealkylation studies in FAB-MS, the porphyria-producing pigment was identified as N -methylprotoporphyrin IX.

High-performance liquid chromatography of N-alkylprotoporphyrins : an investigation of their formation and loss under chemical and enzymatic conditions in vitro

A new technique for resolving N-alkylprotoporphyrins into each structural isomer is described. The technique has been used to investigate the rate of formation and loss of N-alkylporphyrins during reaction of the parent porphyrin with alkyl iodides and to establish the conditions required for optimal yields of the various isomers. Preferential loss of the isomers bearing the N-alkyl group on one of the vinyl-substituted pyrrole rings is observed on prolonged incubation and HI generated during the reaction has been shown to be responsible. A method for detection and partial resolution by HPLC of N-alkylprotoporphyrins produced by liver microsomes in vitro is also described. Microsomes from rats induced with 3-methylcholanthrene produce significantly more N-ethylprotoporphyrin from either 4-ethyl-3,5-diethoxycarbonyl-1,4-dihydro- 2,6-dimethyl-pyridine or ethylhydrazine than do microsomes from control animals, but the isomeric composition of the isolated N-alkylporphyrin differs from that reported in vivo. Evidence that authentic N-alkylporphyrins are lost during incubation with microsomes has been obtained, and here again, the isomers bearing the N-alkyl group on vinyl-substituted pyrrole rings are preferentially lost. Experiments with 14C-labeled N-methylprotoporphyrin show that approximately 40% of the lost porphyrin could be recovered bound covalently to the microsomal pellet.

Isomeric Composition of N -Alkylated Protoporphyrins Produced by Substituted Dihydropyridines in Vivo and in Vitro

Substituted dihydropyridines, like DDC and its 4-ethyl analogue (4-ethyl-DDC)* give rise to N-alkylated porphyrins when given to rats and mice in vivo, by donating their intact 4-alkyl group to one of the pyrrole nitrogens of haem in hepatic cytochrome P-450 (De Matteis et al., 1981; Ortiz de Montellano et al., 1981; Tephly et al., 1981). Because of the asymmetrical arrangement of the two vinyl and propionate side chains in protoporphyrin IX, four structural isomers of N-monoalkylated protoporphyrins are possible, according to which of the four pyrrole nitrogens has been substituted. In a previous paper (De Matteis et al., 1983) we have provided evidence that the isomeric composition of the N-ethyl protoporphyrin produced in vivo by treatment with 4-ethyl-DDC depends on the particular cytochrome P-450 which predominates at the time of treatment, suggesting a role for the apo-cytochrome in directing alkylation preferentially on to one of the pyrrole nitrogens. In this present paper we wish to report additional findings in vivo which are compatible with this interpretation. Production of N-ethyl protoporphyrin is also found in vitro, when isolated microsomes are incubated with 4-ethyl-DDC in presence of NADPH and, although a change in isomeric profile is again found with microsomes containing different cytochrome P-450 enzymes, nevertheless the picture obtained with isolated membranes in vitro is different from that seen in the intact cells in vivo.